Cloning of the Neurodegeneration Gene \u3cem\u3eDrop-Dead\u3c/em\u3e and Characterization of Additional Phenotypes of its Mutation

Mutations in the Drosophila gene drop-dead (drd) result in early adult lethality and neurodegeneration, but the molecular identity of the drd gene and its mechanism of action are not known. This paper describes the characterization of a new X-linked recessive adult-lethal mutation, originally called lot\u27s wife (lwf1) but subsequently identified as an allele of drd (drdlwf); drdlwf mutants die within two weeks of eclosion. Through mapping and complementation, the drd gene has been identified as CG33968, which encodes a putative integral membrane protein of unknown function. The drdlwf allele is associated with a nonsense mutation that eliminates nearly 80% of the CG33968 gene product; mutations in the same gene were also found in two previously described drd alleles. Characterization of drdlwf flies revealed additional phenotypes of drd, most notably, defects in food processing by the digestive system and in oogenesis. Mutant flies store significantly more food in their crops and defecate less than wild-type flies, suggesting that normal transfer of ingested food from the crop into the midgut is dependent upon the DRD gene product. The defect in oogenesis results in the sterility of homozygous mutant females and is associated with a reduction in the number of vitellogenic egg chambers. The disruption in vitellogenesis is far more severe than that seen in starved flies and so is unlikely to be a secondary consequence of the digestive phenotype. This study demonstrates that mutation of the drd gene CG33968 results in a complex phenotype affecting multiple physiological systems within the fly

Isoform- and Cell-Specific Function of Tyrosine Decarboxylase in the Drosophila Malpighian Tubule

The biogenic amine tyramine (TA) is a potent diuretic factor when applied to the Malpighian tubule (MT) of Drosophila melanogaster, stimulating both urine production and transepithelial chloride conductance. Isolated MTs can respond not only to TA but also to its precursor, tyrosine; this observation led to the proposal that MTs are able to synthesize TA from applied tyrosine through the action of the enzyme tyrosine decarboxylase (TDC). In the current study it is shown that the non-neuronal isoform of TDC, Tdc1, is expressed in the principal cells of the MT. A mutant allele of Tdc1, Tdc1f03311, was identified that reduced expression of the mature Tdc1 transcript by greater than 100-fold. MTs isolated from Tdc1f03311 homozygous flies showed no significant depolarization of their transepithelial potential (TEP) or diuresis in response to tyrosine while retaining normal sensitivity to TA. By contrast, a previously identified null mutant allele of the neuronal TDC isoform Tdc2 had no effect on either tyrosine or TA sensitivity. To determine in which cell type of the MT Tdc1 expression is required, flies were generated carrying a UAS-Tdc1 transgene and cell-type-specific Gal4 drivers on a Tdc1f03311 homozygous background. Rescue of Tdc1 expression in principal cells fully restored sensitivity to tyrosine whereas expression of Tdc1 in stellate cells had no rescuing effect. It is concluded that synthesis of TA by Tdc1 in the principal cells of the MT is required for physiological responses to tyrosine. TA synthesis in the MT is the first reported physiological role for Drosophila Tdc1

Characterization of Transepithelial Potential Oscillations in the \u3cem\u3eDrosophila\u3c/em\u3e Malpighian Tubule

The Malpighian tubule of Drosophila melanogaster is a useful model system for studying the regulation of epithelial ion transport. In acutely isolated tubules, the transepithelial potential (TEP) undergoes large oscillations in amplitude with a period of approximately 30s. The TEP oscillations are diminished by reductions in the peritubular chloride concentration in a manner consistent with their being caused by fluctuations in chloride conductance. The oscillations are eliminated by pretreating tubules with the calcium chelator BAPTA-AM, although removal of peritubular calcium has no effect, suggesting that the oscillations are a result of either the release of calcium from intracellular stores or the entry of calcium from the tubule lumen. Transcripts encoding two calcium-release channels, the ryanodine receptor and the inositol trisphosphate receptor, are detectable in the tubule by reverse transcription–polymerase chain reaction. To identify the cell type responsible for the oscillations, tubules were treated with diuretic hormones known to alter calcium levels in each of the two cell types. Leucokinin-IV, which increases calcium levels in the stellate cells, suppressed the oscillations, whereas cardioacceleratory peptide 2b (CAP2b), which increases calcium levels in the principal cells, had no effect. These data are consistent with a model in which rhythmic changes in transepithelial chloride conductance, regulated by intracellular calcium levels in the stellate cells, cause the TEP oscillations

Identification of Multiple Functional Receptors for Tyramine on an Insect Secretory Epithelium

The biogenic amine tyramine (TA) regulates many aspects of invertebrate physiology and development. Although three TA receptor subtypes have been identified (TAR1-3), specific receptors have not been linked to physiological responses in native tissue. In the Malpighian (renal) tubule of Drosophila melanogaster, TA activates a transepithelial chloride conductance, resulting in diuresis and depolarization of the transepithelial potential. In the current work, mutation or RNAi-mediated knockdown in the stellate cells of the tubule of TAR2 (tyrR, CG7431) resulted in a dramatic reduction, but not elimination, of the TA-mediated depolarization. Mutation or knockdown of TAR3 (tyrRII, CG16766) had no effect. However, deletion of both genes, or knockdown of TAR3 on a TAR2 mutant background, eliminated the TA responses. Thus while TAR2 is responsible for the majority of the TA sensitivity of the tubule, TAR3 also contributes to the response. Knockdown or mutation of TAR2 also eliminated the response of tubules to the related amine octopamine (OA), indicating that OA can activate TAR2. This finding contrasts to reports that heterologously expressed TAR2 is highly selective for TA over OA. This is the first report of TA receptor function in a native tissue and indicates unexpected complexity in the physiology of the Malpighian tubule

The MinK Potassium Channel Exists in Functional and Nonfunctional Forms When Expressed in the Plasma Membrane of \u3cem\u3eXenopus\u3c/em\u3e Oocytes

The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. In order to measure the levels of minK protein in the plasma membrane, we have modified the minK gene by inserting a 9 amino acid epitope into the N- terminal domain of the protein sequence. When intact live oocytes are injected with the modified minK RNA and subsequently incubated with an antibody to this epitope, specific binding is detected, indicating that the N-terminal domain is extracellular. We found that when oocytes are injected with amounts of minK mRNA up to 50 ng, the levels of protein at the surface are proportional to the amount of injected mRNA. In contrast, the amplitude of the minK current recorded in the oocytes saturates at 1 ng of injected mRNA. Although the amplitude of the currents is not altered by increasing mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA. In particular, activation is slower with higher levels of minK protein in the plasma membrane. Finally, we find that increasing intracellular cAMP levels, which increases the amplitude of minK currents, does not alter surface expression of the minK protein but produces a small increase in the rate of activation of the current. Our results support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte

Modulation by cAMP of a Slowly Activating Potassium Channel Expressed in \u3cem\u3eXenopus\u3c/em\u3e Oocytes

When expressed in the Xenopus oocyte, the minK protein induces a slowly activating voltage-dependent potassium current (Isk). We studied the modulation of this current by altering intracellular cAMP levels and found that the amplitude of Isk is dramatically increased by treatments that raise cAMP levels and decreased by agents that lower cAMP levels. Preinjection of a protein inhibitor of the cAMP-dependent protein kinase blocked the effects of increased cAMP levels. There were no changes in the voltage dependence or kinetics of Isk. Mutations that eliminate a potential phosphorylation site on the minK protein did not block the effects of activating the kinase. In addition, the membrane capacitance of the oocyte increased and decreased in parallel with Isk. Our results fit a mechanism in which channel proteins are selectively inserted into and removed from the plasma membrane in response to changes in kinase activity

Developmental Expression of \u3cem\u3eDrop-Dead\u3c/em\u3e is Required for Early Adult Survival and Normal Body Mass in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

In Drosophila melanogaster, mutations in the gene drop-dead (drd) result in early adult lethality, with flies dying within 2 weeks of eclosion. Additional phenotypes include neurodegeneration, tracheal defects, starvation, reduced body mass, and female sterility. The cause of early lethality and the function of the drd protein remain unknown. In the current study, the temporal profiles of drd expression required for adult survival and body mass regulation were investigated. Knockdown of drd expression by UAS-RNAi transgenes and rescue of drd expression on a drd mutant background by a UAS-drd transgene were controlled with the Heat Shock Protein 70 (Hsp70)-Gal4 driver. Flies were heat-shocked at different stages of their life cycle, and the survival and body mass of the resulting adult flies were assayed. Surprisingly, the adult lethal phenotype did not depend upon drd expression in the adult. Rather, expression of drd during the second half of metamorphosis was both necessary and sufficient to prevent rapid adult mortality. In contrast, the attainment of normal adult body mass required a different temporal pattern of drd expression. In this case, manipulation of drd expression solely during larval development or metamorphosis had no effect on body mass, while knockdown or rescue of drd expression during all of pre-adult (embryonic, larval, and pupal) development did significantly alter body mass. Together, these results indicate that the adult-lethal gene drd is required only during development. Furthermore, the mutant phenotypes of body mass and lifespan are separable phenotypes arising from an absence of drd expression at different developmental stages

Neurodegeneration in \u3cem\u3eDrop-Dead\u3c/em\u3e Mutant \u3cem\u3eDrosophila melanogaster\u3c/em\u3e Is Associated with the Respiratory System but Not with Hypoxia

Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia

Association between physician characteristics and payments from industry in 2015-2017: observational study.

OBJECTIVE:To investigate the association between physician characteristics and the value of industry payments. DESIGN:Observational study. SETTING AND PARTICIPANTS:Using the 2015-2017 Open Payments reports of industry payments linked to the Physician Compare database, we examined the association between physician characteristics (physician sex, years in practice, medical school attended and specialty) and the industry payment value, adjusting for other physician characteristic and institution fixed effects (effectively comparing physicians practicing at the same institution). MAIN OUTCOME MEASURES:Our primary outcome was the value of total industry payments to physicians including (1) general payments (all forms of payments other than those classified for research purpose, eg, consulting fees, food, beverage), (2) research payments (payments for research endeavours under a written contract or protocol) and (3) ownership interests (eg, stock or stock options, bonds). We also investigated each category of payment separately. RESULTS:Of 544 264 physicians treating Medicare beneficiaries, a total of $5.8 billion in industry payments were made to 365 801 physicians during 2015-2017. The top 5% of physicians, by cumulative payments, accounted for 91% of industry payments. Within the same institution, male physicians, physicians with 21-30 years in practice and physicians who attended top 50 US medical schools (based on the research ranking) received higher industry payments. Across specialties, orthopaedic surgeons, neurosurgeons and endocrinologists received the highest payments. When we investigated individual types of payment, we found that orthopaedic surgeons received the highest general payments; haematologists/oncologists were the most likely to receive research payments and surgeons were the most likely to receive ownership interests compared with other types of physicians. CONCLUSIONS:Industry payments to physicians were highly concentrated among a small number of physicians. Male sex, longer length of time in clinical practice, graduated from a top-ranked US medical school and practicing certain specialties, were independently associated with higher industry payments

Defective Gut Function in \u3cem\u3eDrop-Dead\u3c/em\u3e Mutant \u3cem\u3eDrosophila\u3c/em\u3e

Mutation of the gene drop-dead (drd ) causes adult Drosophila to die within 2 weeks of eclosion and is associated with reduced rates of defecation and increased volumes of crop contents. In the current study, we demonstrate that flies carrying the strong allele drdlwf display a reduction in the transfer of ingested food from the crop to the midgut, as measured both as a change in the steady-state distribution of food within the gut and also in the rates of crop emptying and midgut filling following a single meal. Mutant flies have abnormal triglyceride (TG) and glycogen stores over the first 4 days post-eclosion, consistent with their inability to move food into the midgut for digestion and nutrient absorption. However, the lifespan of mutants was dependent upon food presence and quality, suggesting that at least some individual flies were able to digest some food. Finally, spontaneous motility of the crop was abnormal in drdlwf flies, with the crops of mutant flies contracting significantly more rapidly than those of heterozygous controls. We therefore hypothesize that mutation of drd causes a structural or regulatory defect that inhibits the entry of food into the midgut